High-efficiency retroviral-mediated gene transfer into human and nonhuman primate peripheral blood lymphocytes

B. A. Bunnell, L. M. Muul, R. E. Donahue, R. M. Blaese, R. A. Morgan

Research output: Contribution to journalArticle

234 Scopus citations

Abstract

Peripheral blood lymphocytes (PBLs) are primary targets for gene therapy of inherited and acquired disorders of the immune system. We describe the development of an optimized transduction system that provides for high- efficiency retrovirus-mediated gene transfer into primary PBLs. This optimized transduction protocol combines centrifugation of the lymphocytes (1000 x g) at the inception of transduction with phosphate depletion, low- temperature incubation (32°C), and the use of the packaging cell line PG13. Gene marking studies of human and primate PBLs using these optimized transduction conditions demonstrated that the transduction efficiency exceeded 50% of the total lymphocyte population. The optimized transduction efficiency of PBLs with amphotropic retroviral vectors was in excess of 25%. The transduction procedure does not alter phenotype, viability, or expansion of the transduced cells. Our data indicate that this optimized transduction system leads to high-efficiency gene transfer into primary human lymphocytes, which obviates the requirement for selection of transduced cells prior to gene-therapy procedures. Thus, large quantities of healthy retrovitally transduced lymphocytes containing a broad immunological repertoire can be generated for use in clinical protocols. Our results represent a significant improvement in the methodology for the transduction of lymphocytes for gene therapy.

Original languageEnglish
Pages (from-to)7739-7743
Number of pages5
JournalProceedings of the National Academy of Sciences of the United States of America
Volume92
Issue number17
DOIs
StatePublished - 1995

Keywords

  • adenosine deaminase
  • gene therapy
  • retroviral vector

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